Elucidating the in vivo interactome of HIV-1 RNA by hybridization capture and mass spectrometry

Abstract

HIV-1 replication requires myriad interactions between cellular proteins and the viral unspliced RNA. These interactions are important in archetypal RNA processes such as transcription and translation as well as for more specialized functions including alternative splicing and packaging of unspliced genomic RNA into virions. We present here a hybridization capture strategy for purification of unspliced full-length HIV RNA-protein complexes preserved in vivo by formaldehyde crosslinking, and coupled with mass spectrometry to identify HIV RNA-protein interactors in HIV-1 infected cells. One hundred eighty-nine proteins were identified to interact with unspliced HIV RNA including Rev and Gag/Gag-Pol, 24 host proteins previously shown to bind segments of HIV RNA, and over 90 proteins previously shown to impact HIV replication. Further analysis using siRNA knockdown techniques against several of these proteins revealed significant changes to HIV expression. These results demonstrate the utility of the approach for the discovery of host proteins involved in HIV replication. Additionally, because this strategy only requires availability of 30 nucleotides of the HIV-RNA for hybridization with a capture oligonucleotide, it is readily applicable to any HIV system of interest regardless of cell type, HIV-1 virus strain, or experimental perturbation.

Figure 1

(a) HyPR-MS uses hybridization capture for purification of HIV RNA-protein complexes from cell lysate for protein identification by mass spectrometry. (b) A toehold-mediated capture and release strategy was implemented for sequence specific isolation of RNA-protein complexes. (c) A total of three biological replicates were analyzed. Three capture replicates (Capt. Reps.) were conducted for each biological replicate and were each divided into two mass spectrometric analyses (MS Tech. Reps.). (d) Capture efficiency and specificity for each capture was measured using a RT-qPCR assay specific to HIV RNA. RT-qPCR analysis of HIV RNA relative to GAPDH RNA in the capture and lysate samples measured enrichment of HIV RNA after capture. Error bars in graphs are standard deviations of measurements made for all three biological replicates.

Figure 2

Unspliced HIV RNA protein interactor map. All proteins indicated were identified by HyPR-MS to interact with HIV unspliced RNA (with the exception of Tat). Proteins in rectangles have previously been shown to affect specific stages of HIV replication and are grouped accordingly (colored circles). Proteins in ovals have previously been shown to affect HIV replication but the stage of replication was not determined. These proteins are therefore grouped according to their RNA-process related GO annotation. Proteins shaded in grey have been previously identified to interact with segments of HIV RNA; unshaded proteins are novel unspliced HIV RNA interactors. Virus encoded HIV RNA binders are indicated in the large rectangles; and a line connects them to cellular proteins previously shown to interact with these viral proteins^{,,–,,–,–,–,,–,–,,–}. Supplementary Information Table S3 also lists these interactions.

Figure 3

(a) Diagram of the HIV-1 reporter virus used for functional evaluation of HIV RNA interactors. The RNA sequences coding for the fluorescent reporter proteins, CFP and mCherry, are postioned so that three CFP and one mCherry molecules are expressed following translation from the full length and the completely spliced transcripts, respectively. (b) Quantitative analysis of fluorescence from reporter proteins following target protein siRNA knockdown and 48-hours post HIV infection. (c) Western blot analysis of siRNA knocked-down target proteins. Demonstrates >90% knock down of each protein. Images and quantitative analysis of complete blots are in SI Figure S3.